Process for the preparation of beta-lactam derivatives

ABSTRACT

A process for the preparation of Cefuroxime acid (1), which comprises the following steps: (1) Extraction of deacetyl 7-glutaryl ACA (II) aqueous solution at acid pH with organic solvents (for example according to the procedures disclosed in U.S. Pat. No. 5,801,241); (2) drying the resulting solution while preventing lactonization of the intermediate; (3) carbamoylation of the hydroxymethyl group at the 3-position by reaction with chlorosulfonyl isocyanate or similar products; (7) extraction of the carbamoyl derivative from step 3 with water at neutral pH; (8) enzymatic hydrolysis of the amide at the 7-position of the cephalosporanic ring with glutaryl acylase; (6) acylation of the amino group by condensation with 2-furanyl(sin-methoxyimino)acetic acid chloride or mixed anhydride.

[0001] The present invention generally relates to the field of organicchemistry.

[0002] More particularly, the invention relates to a process for thepreparation of Cefuroxime acid, i.e.(6R,7R)-7-[[2-furanyl(sin-methoxyimino)acetyl]amino]-3-carbamoyloxymethylceph-3-em-4-carboxylic)acid, and the salts thereof, starting from(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-hydroxymethyl-ceph-3-em-4-carboxylicacid (deacetyl 7-glutaryl ACA).

[0003] Cefuroxime acid is a key intermediate for the industrialsynthesis of two third generation cephalosporins, Cefuroxime sodium (forthe injection administration) and Cefuroxime Axetil (for the oraladministration). These molecules are therapeutically valuable thanks totheir effective broad spectrum antibacterial activity againstgram-negative bacterials, in particular in the treatment ofimmunodepressed patients. Their effectiveness is advantageously combinedwith remarkable resistance to β-lactamases.

[0004] The synthesis of Cefuroxime disclosed in U.S. Pat. Nos. 3,966,717and 3,974,153 comprises 8 synthetic steps starting from 7-ACA (7-aminocephalosporanic acid). Such high number of steps, which causes a lowoverall yield, is due to the introduction of two protective groups, thefirst (e.g. thienyl acetyl) on the amine function and the second (e.g.benzhydryl) on the 7-ACA acid function.

[0005] Subsequently, processes starting from 7-ACA have been developed(Wilson, E. M. Chemistry and Industry 1984, 217) which do not involvethe use of protective groups and remarkably reduce the number of steps.In particular, the best process, illustrated in Scheme 1, comprises 3steps:

[0006] 1. Conversion of 7-ACA into deacetyl-7-ACA;

[0007] 2. Acylation of the amino group;

[0008] 3. Carbamoylation of the C-3 alcohol group (Scheme 1).

[0009] It has now surprisingly been found that Cefuroxime can beprepared starting from intermediates of 7-ACA enzymatic synthesiswithout isolating any intermediate.

[0010] The enzymatic synthesis of 7-ACA involves, depending on the usedprocess, an intermediate that can either be glutaryl-7-ACA (U.S. Pat.No. 5,424,196; Bianchi, D., Bortolo, R., Golini, P., Cesti, P. LaChimica e l'Industria 1998, 80, 879), which can be enzymaticallyconverted into deacetyl 7-glutaryl ACA (II), or (II) itself, fromfermentative processes yielding des-Cephalosporin C (U.S. Pat. No.4,533,632).

[0011] The process object of this invention, illustrated in Scheme 2,comprises the following steps:

[0012] 1. Extraction of deacetyl 7-glutaryl ACA (II) aqueous solution atacid pH with organic solvents (for example according to the proceduresdisclosed in U.S. Pat. No. 5,801,241).

[0013] 2. Drying the resulting solution while preventing lactonizationof the intermediate.

[0014] 3. Carbamoylation of the hydroxymethyl group at the 3-position byreaction with chlorosulfonyl isocyanate or similar products.

[0015] 4. Extraction of the carbamoyl derivative from step 3 with waterat neutral pH.

[0016] 5. Enzymatic hydrolysis of the amide at the 7-position of thecephalosporanic ring with glutaryl acylase.

[0017] 6. Acylation of the amino group by condensation with 2-furanyl(sin-methoxyimino)acetic acid chloride or mixed anhydride.

[0018] The process of the invention involves a reduction of the numberof the steps compared with the known processes for the preparation ofCefuroxime, as it requires neither the protection of the carboxyl at the4-position of the cephalosporanic ring nor that of the amino group atthe 7-position nor the recovery of any intermediates, thus causing aremarkable increase in the overall yield directly starting fromdes-Cephalosporin C or Cephalosporin C fermentation broth afterenzymatic deacetylation.

[0019] Furthermore, the process of the invention allows making use ofintermediates, which, contrary to 7-ACA, are particularly stable inaqueous solution.

[0020] The process according to the present invention providesCefuroxime acid or a salt thereof which can be transformed into thecorresponding commercial products Cefuroxime sodium and Cefuroximeaxetil.

[0021] The process described above comprises the preparation of anintermediate, which has to day never been described, namely(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-carbamoyloxy-methyl-ceph-3-em-4-carboxylicacid of formula (III)

[0022] or a salt thereof.

[0023] The steps of the process according to the present invention forthe preparation of Cefuroxime acid starting from deacetyl 7-glutaryl ACA(II) are described in detail hereinbelow.

[0024] 1. Extraction of a deacetyl 7-glutaryl ACA (II) aqueous solutionwith an organic solvent, preferably cyclohexanone (U.S. Pat. No.5,801,241).

[0025] A deacetyl 7-glutaryl ACA aqueous solution, at a concentrationranging from 1 to 20%, is adjusted to pH ranging from 1.0 to 3.0,preferably 1.5, at temperatures ranging from 0 to 15° C., preferablyfrom 0 to 5° C. These conditions prevent degradation of the substrate togive a lactone following condensation of the carboxyl at the 4-positionwith the hydroxyl bound to the methyl at the 3-position. The resultingsolution is added with 0.5 to 2 volumes of an organic solvent,preferably cyclohexanone, and extraction is carried out at temperaturesranging from 0 to 5° C. Phase are separated, then the aqueous phase isback-extracted with 0.5÷1.0 volumes of solvent and the organic phasesare combined.

[0026] 2. The resulting organic solution is brought to temperatures from0 to 15° C., preferably from 0 to 5° C., adjusted to apparent pH rangingfrom 6 to 8, preferably 7, with a solution of triethylamine in theorganic solvent used for the extraction. The resulting mixture isconcentrated in vacuo and at a temperature below 25° C., to obtain asuspension with a water content below 0.5%, which is then processed inthe subsequent step.

[0027] 3. Conversion of deacetyl 7-glutaryl ACA (II) into thecorresponding 3-carbamoyloxymethyl derivative (III), by reaction incyclohexanone with an activated isocyanate, preferably chlorosulfonylisocyanate.

[0028] The suspension isolated at the end of the previous step, having aconcentration ranging from 1 to 10%, is cooled to temperatures rangingfrom −30 to 0° C., preferably −10° C., and added, in small portions,with 1÷5 mols of chlorosulfonyl isocyanate per mol of substrate. Theresulting heterogeneous mixture is then kept at such temperature untilcompletion of the reaction, whose progression is monitored by HPLCchromatography.

[0029] 4. Extraction of the carbamoyl derivative obtained in step 3. Thesolution from step 3 is added with 0.1÷0.3 volumes of cold water and theresulting heterogeneous mixture is then adjusted to pH ranging from 6 to8, preferably 7, at temperatures ranging from 0 to 15° C., preferablyfrom 0 to 5° C., with an aqueous ammonia solution. The two phases areseparated; the organic phase is back-extracted with water, to 0.2volumes; the aqueous phases are combined and the resulting solution,having a concentration ranging from 5 to 30%, is processed in thesubsequent step.

[0030] 5. Conversion of the 7-glutaryl 3-carbamoyloxymethyl derivative(III) into the corresponding 7-β-amino derivative (IV) by enzymatichydrolysis of the amide at the 7-position of the cephalosporanic ringwith glutaryl acylase. The resulting solution from the previous step isadded with a glutaryl acylase isolated from an Escherichia Coli culture,suitably supported on a macroreticular resin, preferably polyacrylicepoxide, and the resulting suspension is kept at pH 7.0-9.0, preferably7.5, at temperatures ranging from 20 to 30° C., preferably 25° C., untilcompletion of the reaction. The progression of the hydrolysis of theglutaryl derivative to the corresponding cephalosporanic ring ismonitored by HPLC chromatography. The reaction yield is higher than 85%.After completion of the reaction, the enzyme is removed and the productis converted into Cefuroxime acid.

[0031] 6. (6R,7R)-7-Amino-3-carbamoyloxymethylceph-3-em-4-carboxylicacid (IV) is directly converted into Cefuroxime acid (I), adding theaqueous solution with a concentrated methylene chloride solution of2-furanyl (sin-methoxyimino)-acetic acid chloride or mixed anhydride(preferably the chloride).

[0032] Optionally, intermediate (IV) can be recovered by acidificationto isoelectric pH of the solution from the previous step, then be workedin the acylation of the amino group at the 7-position of thecephalosporanic ring, by following the method described in example 2 ofU.S. Pat. No. 3,974,153.

EXAMPLE 1

[0033] 300 ml of a 6% deacetyl 7-glutaryl ACA aqueous solution, cooledto 0÷5° C., is added with the necessary amount of sulfuric acid toadjust pH to 1.5. The resulting solution is extracted with one volume ofcyclohexanone. The organic phase, kept at a 0÷5° C., is added with thetriethylamine amount necessary to adjust pH to 6.0. The solution is thenconcentrated to water content below 0.5. At the end of the operation,350 ml of a glutaryl deacetyl 7-ACA suspension containing 98% of theactivity present in the solution resulting from extraction at acid pH,are obtained. The resulting solution is then transformed in thesubsequent step.

EXAMPLE 2

[0034] The mixture from the previous step is cooled to −10° C. and thenadded with 13 ml of chlorosulfonyl isocyanate, preventing temperaturefrom exceeding −10° C. After completion of the addition of the reactive,the mixture is kept at −10° C. until the starting product disappears. Atthe end of the synthesis, the reaction yield is checked to be about 95%.150 ml of cold water are added. The heterogeneous mixture is kept at a0÷5° and its pH is adjusted with a 10% sodium carbonate solution. Thetwo phases are separated, the organic phase is back-extracted with 50 mlof water. The resulting solution is then worked in the subsequent step.

[0035] The product from the above step was recovered adjusting to pH=2the final aqueous solution saturated with sodium chloride. The isolatedrecovered was then characterized by analysis of the data from spectraMass and NMR spectrometry.

[0036]¹H-NMR:

[0037] 1.82 ppm, qui, J=7Hz, 2H, side chain CH₂; 2.3 ppm, m, 4H, sidechain methylenes; AB system at 3.4 ppm, methylene at the 2-position;system at 2.3 ppm, methylene bound to the oxygen; 5.05 ppm, d, J=5Hz,1H, H-6; 5.55 ppm, d, J=5Hz, 1H, H-7.

[0038]¹³C-NMR:

[0039] 22.81 ppm (CH₂); 25.92 ppm (CH₂); 35.61 ppm (CH₂); 36.99 ppm(CH₂); 57.98 ppm (CH); 58.90 ppm (CH); 65.84 ppm (CH₂—OR); 117.14 ppm(C-2); 132.04 ppm (C-3); 154.69 ppm (—OCON—); 165.78 ppm (two amideC═O); 169.30 ppm (COO—); 177.73 ppm (COOH).

[0040] Electrospray ESI:

[0041] m/z 388; m/z 410; m/z 344; m/z 327; m/z 349; m/z 309; m/z 299;m/z 281; m/z 253; m/z 185; m/z 172.

[0042] Desadsorption chemical ionization (DCI):

[0043] m/z 233; m/z 214; m/z 216

EXAMPLE 3

[0044] The resulting aqueous solution is diluted with 300 ml of waterand added with 40 g of glutaryl 7-ACA acylase, whose preparation isdescribed in example 12 of U.S. Pat. No. 5,424,196. The heterogeneousmixture stirred at 20° C. until completion of the reaction,automatically adjusting pH to 7.5 with 5% ammonia. The maximumconversion takes place after 60′, when the solution yield is about 86%.The enzyme is removed and the resulting solution is processed in thesubsequent step.

EXAMPLE 4

[0045] A mixture of phosphorous pentachloride (10 g) and methylenechloride (100 ml), in a reactor of suitable volume, cooled at 0° C., isadded with 18.4 ml of N,N-dimethylacetamide. The resulting solution iscooled, added with of 8.5 g of 2-furanyl(sin-methoxyimino)acetic acidand stirred at −10° C. for 15′; 22 ml of cold water are added thereto,keeping the resulting heterogeneous mixture stirred, the two phases areseparated and the organic phase is slowly added to the solution of thesubstrate isolated in the previous step (example 3), suitably cooled,automatically adjusting pH with 10% sodium hydroxide. The resultingmixture is kept at 0° C. until the substrate disappears. The finalmixture is added with 50 ml of N,N-dimethylacetamide, 50 ml ofacetonitrile and 300 ml of water, pH is adjusted to 2 with 2Nhydrochloric acid and the resulting mixture is stirred at 0÷5° C. forone hour, filtered, washed with water, and dried under-vacuum at 30÷35°C. for 8 hours. 15 g of Cefuroxime acid are obtained in an 85% yield.

EXAMPLE 5

[0046] The product from the step described in example 3 is isolated byadjusting to the isoelectric pH the aqueous solution obtained at the endof this step.

[0047] A mixture of 8.4 g of said product, i.e. of (6R,7R)-7-amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylic (IV) acid, 37 mlof N,N-dimethylacetamide, 37 ml of acetonitrile, 21 ml of triethylamineand 5 ml of water is stirred at 0÷2° C. until complete dissolution ofthe product. Separately, a suspension of 7.7 g of phosphorouspentachloride in 75 ml of methylene chloride is added at 0÷2° C. with14.2 ml of N,N-dimethylacetamide and the resulting solution is added at−10° C. with 6.5 g of 2-furanyl (sin-methoxyimino)acetic acid; theresulting solution is added with 17 ml of cold water, keeping themixture at 0÷2° C. for about 15′. After that, the two phases areseparated and the organic phase is added to the substrate solutionprepared above, suitably cooled at −5° C. The reaction mixture is keptat 5÷10° C. until the substrate disappears. The final mixture is addedat 0÷5° C. to a mixture of 570 ml of water and 50 ml of 2N hydrochloricacid, pH is adjusted to=2 with 2N hydrochloric acid and the resultingmixture is stirred at 0÷5° C. for about an hour, then filtered, washedwith water and dried at 30÷35° C. for 8 hours. 11 g of Cefuroxime acidare obtained in an 86% yield.

1. A process for the preparation of Cefuroxime acid (I)

starting from deacetyl 7-glutaryl ACA (II), which comprises thefollowing steps: a) extracting a deacetyl 7-glutaryl ACA (II) aqueoussolution at pH 1-3 and at a temperature of 0-15° C. with an organicsolvent, to obtain an organic phase containing deacetyl 7-glutaryl ACA(II)

b) adjusting said organic phase to pH 6-8, then drying such phase bydistillation under vacuum at a temperature below or equal to 25° C.; c)reacting deacetyl 7-glutaryl ACA (II) with an activated isocyanate attemperatures ranging from −30° to 0° C., to obtain(6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-carbamoyloxymethyl-ceph-3-em-4-carboxylicacid (III)

d) extracting said compound (III) from the reaction mixture with waterat pH of 6-8 and at a temperature of 0-15° C.; e) transforming compound(III) into (6R,7R)-7-amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylicacid (IV)

 by glutaryl acylase enzymatic hydrolysis, at pH 7-9 and at atemperature of 20-30° C.; f) condensing compound (IV) with a2-furanyl(sin-methoxyimino)acetic acid halide or anhydride, to obtainthe desired Cefuroxime acid (I).
 2. A process as claimed in claim 1, inwhich said organic solvent is cyclohexanone.
 3. A process as claimed inclaim 2, in which said deacetyl 7-glutaryl ACA (II) aqueous solution hasa concentration of 1-20%.
 4. A process as claimed in any one of theabove claims, in which in step b) the organic phase is dried to a watercontent below 0.5%.
 5. A process as claimed in any one of the aboveclaims, in which said activated isocyanate is chlorosulfonyl isocyanate.6. A process as claimed in any one of the above claims, in which saidglutaryl acylase is supported on a macroreticular resin.
 7. A process asclaimed in claim 6, in which said resin is a polyacrylic epoxide resin.8. A process as claimed in claim 7, in which said glutaryl acylase isisolated from an Escherichia Coli culture.
 9. The compound of formula(III)

and the salts thereof.